Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

A novel transcribed repeat element from Entamoeba histolytica.

We have identified an unusual 0.55-kb DNA repeat element specific to Entamoeba histolytica (Eh) which we call interspersed element (IE). The IE is a common feature in independently isolated genomic and cDNA fragments. Hybridization of labeled IE sequences to trophozoite DNA, RNA and first-strand cDNA prepared from poly(A)-enriched mRNA indicate that the IE are reiterated about 500 times per Eh trophozoite and that one or more can be found as RNA transcripts. These features and the degree of conservation of IE suggest a possible role for these sequences.

Transient expression of luciferase in Entamoeba histolytica driven by the ferredoxin gene 5' and 3' regions.

We have successfully transfected Entamoeba histolytica trophozoites with constructs containing ferredoxin sequences fused to the reporter gene luciferase. We have determined the conditions and parameters necessary to maximise transient luciferase expression in our system. Our optimal construct gave values of 268 x 10(3) relative light units per second (RLU s-1) when assayed representing a stimulation of 18,000-fold over the control vector. Comparison of differing constructs allowed us to conclude that the 5' and 3' ferredoxin sequences are both necessary for optimal luciferase expression from our vectors. Transcriptional initiation occurs within the consensus sequence ATTCA in both construct and chromosomal ferredoxin promoters.

Influence of bacteria on electrophoretic proteinase patterns of Entamoeba histolytica isolates.

Gelatin SDS- polyacrylamide gel electrophoresis was used to compare proteinase banding patterns under reducing conditions from whole cell lysates of four axenic and four xenic pathogenic strains of Entamoeba histolytica. All strains shared major bands in the 34 and 66-68 kDa regions, whereas only the axenic strains produced major bands at 26, 28, 30 and 45 kDa. One axenic strain, NIH 200, when reassociated with mixed bacterial flora, reverted to an electrophoretic banding pattern characteristic of other xenic strains. These results suggest that the 26-30 kDa and 45 kDa proteinases of E. histolytica are induced by bacterial starvation while others are constitutively expressed. It is also proposed that the axenic bands of 45 and 34 kDa represent respectively, the reduced forms of the 56 and 40 kDa bands reported elsewhere under non-reducing conditions.

Detection of Trichomonas vaginalis antigen in women by enzyme immunoassay.

An enzyme immunoassay (EIA) was developed for the detection of Trichomonas vaginalis antigen in vaginal swabs. Four hundred and eighty two women attending a sexually transmitted disease (STD) clinic were tested; 44 (9.1%) were positive by culture, 32 (6.6%) were positive by wet film examination, and 54 (11.2%) were considered to be positive for trichomonal antigen by EIA. Taking culture as the reference method, the EIA had a sensitivity of 93.2% and a specificity of 97.5%. The predictive value of a positive test was 82% and that of a negative test was 99.3%.

New rapid latex agglutination test for diagnosing Trichomonas vaginalis infection.

A newly developed latex agglutination test for Trichomonas vaginalis infection was compared for sensitivity, specificity, efficiency, and positive and negative predictive values with microscopy, culture, and an enzyme linked immunosorbent assay (ELISA) in the diagnosis of 395 women attending a genitourinary medicine clinic. T vaginalis infection was diagnosed in 42 (11%) women. The sensitivities of both the latex agglutination test and the ELISA were 95% compared with 74% for microscopy and 76% for culture. The latex test was specific and showed no cross reaction with a wide range of other genital tract infections. The latex agglutination test can detect antigen in both soluble and insoluble forms, and as it is simple to perform, can be undertaken during routine examination without recourse to special equipment or training. Further evaluation is required.

Enzyme-linked immunosorbent assay for the detection of anti-Giardia specific immunoglobulin G in filter paper blood samples.

Conventional diagnosis of infection with Giardia duodenalis is by faecal examination but the sensitivity of a single examination is low. Serological tests, although not always positive, are more acceptable to patients and are useful in determining the prevalence of giardiasis in large populations. We show here that blood collected on filter paper and dried provides an excellent source of material for enzyme-linked immunosorbent assays; using samples from a group of 88 stool-negative and 45 positive patients, the optimum results were obtained by taking the control mean optical density plus one standard deviation as the negative/positive cut-off value. The sensitivity was 91% (2/45 false negatives), and the specificity 95% (4/88 false positives). This method should be particularly useful for large-scale surveys in developing countries or wherever serological testing is done in central laboratories.

The effect of axenic versus xenic culture conditions on the total and secreted proteolytic activity of Entamoeba histolytica strains.

Proteolytic activity was measured in lysates of four axenic and five xenic cultures of different strains of pathogenic E. histolytica, and in five non-pathogenic strains ("E. dispar") growing in xenic culture. There was no significant difference in total proteolytic activity, using either gelatin or albumin as substrate, between the two sets of xenic cultures; the axenic pathogens however had significantly higher levels of activity than the xenic pathogens, the levels appearing to correlate with virulence. An axenic strain (NIH 200) reassociated with mixed bacterial flora reverted to close to xenic levels of activity. There was no evidence of secretion of an elevated proportion of the total enzymic activity by pathogenic strains, and expression levels may owe more to culture conditions than to genotype.

A DNA probe specific to pathogenic Entamoeba histolytica.

A DNA sequence, IE-gen1 (3.1 kb), was isolated from the pathogenic strain of E. histolytica NIH-200. IE-gen1 was identified by the subtractive hybridization of a genomic library to a cDNA probe prepared from NIH-200 trophozoites. The IE-gen1 probe specifically detected pathogenic E. histolytica in slot blots of genomic DNA and Northern blots, but not other Entamoeba species and additional human parasites. This genomic probe could detect with complete specificity DNA from about 10(3) organisms. The IE-gen1 probe could be related to highly specialized loci in pathogenic E. histolytica, and is likely to be a valuable DNA reagent for clinical diagnosis and epidemiological investigations.

Gut Coccidia--Isospora, Cryptosporidium, Cyclospora and Sarcocystis.

The gut Coccidia are members of a large, varied, and exclusively intracellular group of protozoan parasites, four species of which (Isospora, Cryptosporidium, Cyclospora, and Sarcocystis) are human pathogens. The first three, but particularly Cryptosporidium parvum, have moved from medical curiosities to major problems with the coming of the acquired immunodeficiency syndrome (AIDS) epidemic, but are now known to also cause disease in the immunocompetent patient. They are easy to acquire and difficult to remove from the environment and, in the case of cryptosporidiosis, impossible to treat properly. Further research into many aspects of the biology of these organisms is urgently needed.

The amoeba enigma.

After more than 70 years of intermittent debate over the true relationship between the 'pathogenic' and 'non-pathogenic' forms of Entamoeba histolytica, the application of molecular biology has finally yielded an unambiguous answer: these are not interconvertible phenotypes of the same parasite, a kind of unicellular Jekyll and Hyde, but two quite distinct genetic entities that just happen to look the same. But given the overwhelming evidence now available from gene sequences, pointing to an evolutionary divergence some tens of millions of years ago, why is it that certain eminent workers in the field are still claiming that, at least in vitro, conversion between the two phenotypes can take place? In this article Bill Spice and John Ackers review recent developments in the molecular biology of E. histolytica and assess the continuing controversy over the status of this enigmatic parasite.

Urine culture for the detection of Trichomonas vaginalis in men.

Urine samples were collected from 248 men, 21 of whom were known contacts of women infected with Trichomonas vaginalis. This organism was cultured from only three of the 21 specimens from patients in the contact group. The cultural technique was shown to be capable of reliably detecting small numbers of organisms under practical conditions; it appears, therefore, that most male contacts shed relatively few trichomonads and that the infective dose for women must be correspondingly small.

Taxonomic distribution of the antigen eliciting bactericidal antibody for Bordetella pertussis.

Strains of Bordetella pertussis varied in their ability to elicit (in mice) an antibody bactericidal for an antiserum-sensitive strain of B. pertussis, although antibody was usually detectable after only one injection. High titres were produced by a course of seven injections with all strains of B. pertussis tested (six of phase I and three of phase IV) but not with three strains of other Bordetella species nor with two unrelated organisms, a finding of possible taxonomic value. Preliminary investigations have not revealed whether strain vaiations are due to quantitative or qualitative differences in either the bacterial lipopolysaccharide or the carrier protein necessary for antibody production, or whether they may be due to differences in heat lability of 'bactericidal antigen'.